CN109554445B - Effective and simple method for analyzing genetic relationship between peanut species - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to an effective and simple method for analyzing genetic relationship among arachnids. Specifically, a high mutation region is obtained by screening peanut chloroplast genome, and the genetic relationship among peanut species is analyzed by simple amplification and sequencing. The method comprises the following steps: (1) obtaining a chloroplast whole genome gene sequence of cultivated peanuts and related species thereof according to an existing database; (2) comparing and analyzing gene sequences of the chloroplast whole genomes of a plurality of species, anchoring high mutation regions in the chloroplast whole genome range and annotating; (3) and (3) designing a primer for the selected high mutation region, and constructing a phylogenetic tree after PCR amplification and first-generation sequencing. The invention utilizes the psbE-petL of the high mutation region of the arachis, can effectively analyze the genetic relationship among the arachis species through simple amplification and paper mulberry, saves time, labor and cost, is a great breakthrough in effectively analyzing the genetic relationship among the arachis species, and has outstanding practical significance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an effective and simple method for analyzing genetic relationship among arachnids.
Background
The peanut cultivated in the peanut cultivation method has wide planting area, but relatively narrow genetic base. Therefore, it is very important to widen the genetic basis of germplasm resources of the cultivated peanuts. The peanut contains 81 named species, is rich in wild germplasm resources and excellent in resource genes, and has wide adaptability in tropical and subtropical climates. Compared with the cultivated peanut, the wild germplasm has disease and pest resistant genes such as leaf spot, rust disease, bacterial wilt and the like, and is an important gene source for improving peanut varieties. Although some germplasm resources have no cultivation value, the method can obtain new species or achieve the aim of improving the existing cultivated species by utilizing favorable genes of the germplasm resources through hybrid breeding according to different purposes. The method discloses the genetic relationship between the cultivated peanuts and other wild species of the peanut block, and is an important reference for introducing wild resources and storing genetic resources. In recent years, genomics analysis and research has been promoted with the development of high throughput sequencing technologies. However, the analysis of the phylogenetic relationship among arachnids through the chloroplast whole genome sequence requires multiple steps of library construction, sequencing, raw data filtering, assembly, big data analysis and the like, and finally the construction of the phylogenetic tree based on the chloroplast whole genome sequence is completed. The above method is time-consuming and expensive, and bioinformatics analysis is complicated. Therefore, a method for analyzing the genetic relationship among peanut species, which is simple, short in time, high in efficiency and low in cost, is urgently needed.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a method for analyzing the genetic relationship among peanut species, which is simple, short in time, high in efficiency and low in cost.
The technical scheme of the invention is as follows:
an effective and simple method for analyzing the genetic relationship between peanut species utilizes the screening of peanut chloroplast genome to obtain a high mutation region, and can quickly and effectively analyze the genetic relationship between peanut species through simple amplification and sequencing.
The effective and simple method for analyzing the genetic relationship among peanut species comprises the following steps:
(1) acquiring the whole genome gene sequence of chloroplast of the peanut of the cultivar and the related species thereof according to the record of the existing database;
(2) carrying out nucleotide polymorphism analysis on the gene sequences of the whole chloroplast genome of multiple species, anchoring high mutation regions in the whole chloroplast genome range, and annotating;
(3) and (3) designing a primer for the selected high mutation region, carrying out PCR amplification and first-generation sequencing, and constructing a phylogenetic tree to analyze the genetic relationship among peanut species.
Further, in the step (2), a nucleotide diversity alignment of different windows through calculation of a 500bp size sliding window is used to anchor a high nucleotide diversity domain on the chloroplast whole genome using VISTA software.
Further, the high mutation region is a psbE-petL intergenic region, the sequence of the psbE-petL intergenic region is shown in a sequence table 41, and the psbE-petL intergenic region is DNA barcode of Arachis.
Further, in the step (3), in the process of designing the primer, the primer design is performed on the fragment length of about 500bp of the total length of the high mutation region.
Furthermore, the upstream design range of the primer is 1-100bp, the downstream design range is 600-700bp, the size of the product is 500-700bp, and the annealing temperature is 58 ℃.
Further, in the step (3), randomly selecting a plurality of peanut species with chloroplast complete genome, randomly selecting 2-3 complete seeds from each species by a DNA extraction method, soaking the seeds in a culture dish for 3-4 days, placing the seeds into a culture box after the seeds germinate and sprout, culturing at 28 ℃ for 10-13 days, and extracting the DNA of peanut leaves by using a plant genome DNA extraction kit.
Further, in the step (3), the reaction system for PCR amplification is Master Mix 12.5 μ L, DNA template 0.5 μ L, ddH2O10. mu.L, forward primer 1. mu.L, and reverse primer 1. mu.L.
Further, in the step (3), the PCR amplification reaction conditions are pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 50s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 50s, 33 cycles, and re-extension at 72 ℃ for 7 min.
Further, the amplified fragment sequence was obtained by a one-generation sequencing method.
Further, in the step (3), a phylogenetic tree is constructed for the plurality of amplified sequence segments by using MEGA software.
The invention achieves the following beneficial effects:
according to the invention, the special chloroplast genome high mutation region psbE-petL among all species of arachis, namely the DNA barcode of arachis, is anchored, and the constructed phylogenetic tree is compared and analyzed with the phylogenetic tree constructed through chloroplast complete genes, so that a relatively consistent result is obtained. Therefore, the high mutation region psbE-petL of the peanut chloroplast genome is proved to be used for analyzing the genetic relationship among peanut species. In addition, the invention can effectively analyze the interspecific genetic relationship by screening the peanut chloroplast genome high mutation region and only by simple amplification and comparison, thereby saving time, labor and cost, being a great breakthrough in analyzing the interspecific genetic relationship of peanuts and having outstanding practical significance.
Drawings
FIG. 1 is a phylogenetic tree constructed using highly mutated regions of the chloroplast genome.
FIG. 2 is a phylogenetic tree constructed using the chloroplast genome-wide.
Detailed Description
To facilitate understanding of the invention by those skilled in the art, specific embodiments of the invention are described below with reference to examples.
Examples
1 big data analysis
Biological types of 4 cultivated peanuts (GenBank accession No. MG814006 for var. rustigiata Waldron, MG814007 for var. hirsute Kohler, MG814008 for var. hypogaea L. and MG814009 for var. vulgar Harz) and chloroplast whole genomes of 11 wild species (MK144818 for A. monicola, MK144823 for A. paraguariensis, MK144822 for A. duranensis, MK144819 for A. stearosperma, MK144820 for A. basizocoi, MK144824 for A. carrenasiansis, MK144826 for A. helodedes, MK for A. corrigena, MK 144144821 for A. 144821 for MK. rusticans, MK144828 for A. corrigena. 144144821 for MK. cori, MK for MK. 14414414414414414413 for A. cori for MK. sylvestris, MK for A. cori, MK for A. moras, MK.
And MK144825 for A. villosa) sequence, and obtaining candidate high mutation regions through comparison and analysis of whole genome sequences. Specifically, the VISTA software was used to anchor the highly mutated region on the chloroplast genome by nucleotide diversity alignment with a 500bp size sliding window.
2 verification experiment
2.1 investigation materials
4 parts of cultivated peanuts are selected, and 11 parts of wild peanut seeds are selected.
2.2 design of primers
A high mutation region at the whole genome level of peanut chloroplast, namely a psbE-petL intergenic region, is obtained by bioinformatics analysis of high-throughput data, and is shown in a sequence table 41. The primers are designed at two ends of the region, and the total length is 593 bp. Primer premier6.0 is used for primer design, the upstream design range is 1-100bp, the downstream design range is 600-700bp, the size of a product is 593bp, and the annealing temperature is 58 ℃. The primer synthesis information is shown in Table 1.
TABLE 1 primer Synthesis information
2.3 extraction of DNA
Randomly selecting 2-3 complete seeds from each germplasm material, soaking the seeds in a culture dish for 3-4 days, placing the seeds into a culture box after the seeds germinate and sprout, and culturing at 28 ℃ for 10-13 days. Peanut leaf DNA was extracted using a plant genomic DNA extraction kit (TIANGEN, Beijing). After gel electrophoresis detection (US Everbright Inc, Suzhou, China) using Super GelRed (S-2001), DNA integrity and purity were determined using a NanodropTM2000(Thermo Scientific) spectrophotometer. 4 DNA samples of the obtained cultivated species and 11 DNA samples of the wild species, wherein each DNA sample ensures that the unit concentration is not less than 50 ng/mu L and the total mass is more than 2 mu g, and the DNA samples are stored in a refrigerator at minus 80 ℃ for later use.
2.4PCR amplification and electrophoresis detection
The genomic DNA sample was amplified using primer number 20, the total volume of the reaction system was 25. mu.L, containing Master Mix 12.5. mu.L, DNA template 0.5. mu.L, ddH2O10. mu.L, forward primer 1. mu.L, and reverse primer 1. mu.L. The reaction conditions include pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 50s, annealing at 60 deg.C for 1min, extension at 72 deg.C for 50s, 33 cycles, re-extension at 72 deg.C for 7min, and storage at 4 deg.C. The amplification product was electrophoresed with 1% agarose gel at 120V, electrophoresis 40miAnd n, sequencing the clear bands after detection by a gel imaging analysis system (Shanghai energy).
2.5 sequencing and data analysis
The nucleotide polymorphisms of each sliding window were counted by one-generation sequencing, MEGAv7.0((http:// www.megasoftware.net) and aligned analysis, the sequence insertion and deletion data and the pi values (nucleic acid diversity), theta values (nucleic acid polymorphism) and other related statistical data were counted by DNAsp software as shown in Table 2, phylogenetic trees were constructed by MEGA software, and the evolutionary relationships among species were analyzed as shown in FIG. 1.
Table 2 insertion and deletion statistics of sequences
Comparative example
Systemic relation paper mulberry by chloroplast whole genome sequence
1. Extraction of DNA (same example)
2. Library construction and sequencing
By creating a two-terminal library (Illumina, China) with an average insert size of 350bp, different linkers were added to different species of the library. The constructed library was first assessed for quality on a Caliper LabChip GX using a high sensitivity detection kit (Caliber, usa), then hybridized and amplified on a flow cell, followed by generation of clonal clusters on the cBot platform using Truseq PE clusterikit v3-cBot-HS (Illumina, china). High-throughput sequencing was performed on the Illumina Hiseq Xten platform using the Truseq v3-HS kit (Illumina, china).
3. Raw data filtering
Raw reads greater than 1Gb data volume were generated for each sample by Illumina sequencing. These raw reads were analyzed using NGS QC ToolKit v2.3.3, filtering out low quality data and removing the adaptor sequence, with the reads length and phred values set to 80 and 30, respectively. And, the filtered reads were aligned to the cultivar peanut reference genome using the software bowtie (GenBank accession No. kx257487, Prabhudas et al.2016). Next, high quality reads belonging to the chloroplast genome were extracted and assembled into contigs using the software SPAdes v3.9.0(Bank evich et al, 2012) (different k-mer sizes: 93,105,117and 121). These contigs will be further assembled into a complete chloroplast genome by the NOVOPlasty v2.6.2 software (Dierckxsens et al, 2016).
4. Tree building
Phylogenetic trees were constructed using MEGA software, and phylogenetic relationships between species were analyzed as shown in FIG. 2.
Analysis of the results of examples and comparative examples:
as shown in FIGS. 1 and 2, the phylogenetic tree constructed by the mutation region is more consistent with the phylogenetic tree between species constructed by the chloroplast genome complete sequence of the corresponding species. Thus, it was confirmed that genetic relationship analysis between Arachis species can be accomplished simply by amplifying the highly mutated region (psbE-petL intergenic region) and constructing a tree.
The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Sequence listing
<110> institute for peanut research in Shandong province
<120> effective and simple method for analyzing genetic relationship between peanut species
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tagaaagaag aaatagaaat attctagaaa ctagaaataa aaaaataatt tgaaattctt 240
tttggattat tttactcaac ttacgagttg ctcaaaaaat ctgttgattt tgaaccacag 300
gatggatagt tgtcataggt gatgaattga tttcttaccg atgtcactct atttttgttc 360
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ttcttctcct attcgattgt atttccttgg aaac 454
Claims (5)
1. An effective and simple method for analyzing genetic relationship among peanut species is characterized in that: screening chloroplast genomes of a plurality of species of arachis to obtain a high mutation region, and rapidly and effectively analyzing the genetic relationship among the species of arachis through simple amplification and sequencing; the method comprises the following steps:
(1) acquiring the whole genome gene sequence of chloroplast of the peanut of the cultivar and the related species thereof according to the record of the existing database;
(2) carrying out nucleotide polymorphism analysis on the gene sequences of the whole chloroplast genome of multiple species, anchoring high mutation regions in the whole chloroplast genome range, and annotating;
(3) after primer design, PCR amplification and first-generation sequencing are carried out on the selected high mutation region, a phylogenetic tree is constructed to rapidly and effectively analyze the ArachisAn interspecies genetic relationship; the high mutation region ispsbE-petL intergenic region, amplifying the samepsbE-petThe nucleotide sequences of the upstream primer and the downstream primer of the L gene spacer region are as follows:
an upstream primer: 5'-GGGCATTCTAAGGTAACTCGTT-3', respectively;
a downstream primer: 5'-AAGCACTTCCCTAAGTTTCCAA-3' are provided.
2. The method of claim 1, wherein the method comprises the steps of: in the step (3), several peanut species with chloroplast complete genome are randomly selected, 2-3 complete seeds are randomly selected from each species by a DNA extraction method, the seeds are soaked in a culture dish for 3-4 days, the seeds are placed in a culture box after sprouting and budding, the seeds are cultured at 28 ℃ for 10-13 days, and the DNA of peanut leaves is extracted by using a plant genome DNA extraction kit.
3. The method of claim 1, wherein the method comprises the steps of: in the step (3), the reaction system for PCR amplification is 0.5 mu L, ddH2O10 mu L of Master Mix 12.5 mu L, DNA template, 1 mu L of upstream primer and 1 mu L of downstream primer.
4. The method of claim 1, wherein the method comprises the steps of: in the step (3), the PCR amplification reaction conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 50s, annealing at 58 ℃ for 1min, extension at 72 ℃ for 50s, 33 cycles and re-extension at 72 ℃ for 7min, and the amplified fragment sequence is obtained by a one-generation sequencing method.
5. The method of claim 1, wherein the method comprises the steps of: in the step (3), a system relationship tree is constructed for the plurality of amplified sequence segments by using MEGA software.
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